Metaproteomics provides functional insight into activated sludge wastewater treatment

Abstract

Background: Through identification of highly expressed proteins from a mixed culture activated sludge system this study provides functional evidence of microbial transformations important for enhanced biological phosphorus removal (EBPR).

Methodology/principal findings: A laboratory-scale sequencing batch reactor was successfully operated for different levels of EBPR, removing around 25, 40 and 55 mg/l P. The microbial communities were dominated by the uncultured polyphosphate-accumulating organism "Candidatus Accumulibacter phosphatis". When EBPR failed, the sludge was dominated by tetrad-forming alpha-Proteobacteria. Representative and reproducible 2D gel protein separations were obtained for all sludge samples. 638 protein spots were matched across gels generated from the phosphate removing sludges. 111 of these were excised and 46 proteins were identified using recently available sludge metagenomic sequences. Many of these closely match proteins from "Candidatus Accumulibacter phosphatis" and could be directly linked to the EBPR process. They included enzymes involved in energy generation, polyhydroxyalkanoate synthesis, glycolysis, gluconeogenesis, glycogen synthesis, glyoxylate/TCA cycle, fatty acid beta oxidation, fatty acid synthesis and phosphate transport. Several proteins involved in cellular stress response were detected.

Conclusions/significance: Importantly, this study provides direct evidence linking the metabolic activities of "Accumulibacter" to the chemical transformations observed in EBPR. Finally, the results are discussed in relation to current EBPR metabolic models.

Figure 1. Representative FISH micrographs of the activated sludges analysed in this study.

(A) EBPR₂₈ sludge, (B) EBPR₄₂ sludge, (C) EBPR₅₅ sludge and (D) nEBPR₇₀ sludge. Cells detected with probe EUBMIX only are green (A, B, C and D). Cells detected with both EUBMIX and PAO651 probes (A, B and C) and cells detected with both EUBMIX and ALF1b probes (D) are yellow-orange. Highlighted area in pane d corresponds to magnified region hybridised only with the ALF1b probe in the top right hand corner. Images taken under the different excitation wavelengths for CY3 and FITC were combined using Adobe Photoshop. Cells were observed under x 630 magnification, bars = 10 µm.

Figure 2. Representative 2D-PAGE separations of proteins extracted from (A) the EBPR₂₈ sludge, (B) the EBPR₄₂ sludge, (C) the EBPR₅₅ sludge and (D) the nEBPR₇₀ sludge.

Approximate protein molecular mass ranges are provided on the left and isoelectric point ranges are provided on the bottom of the gel images.

Figure 3. Master 2D-PAGE gel of the EBPR matchset with excised protein spots highlighted.

Spot numbering corresponds to the numbering used in Table 2, and supporting information Table S3.

Figure 4. Proposed metabolic model for the (A) anaerobic and (B) aerobic phase of EBPR inferred from the proteomic data.

Identified proteins catalysing individual reactions are highlighted in green [best MASCOT metagenomic sequence match located on a scaffold source binned as “A. phosphatis”, i.e. strong association with the “A. phosphatis” composite genome], orange [best MASCOT sequence match located on a scaffold source binned as “other Accumulibacter” for which a strong BLAST hit (>90 % identity) was obtained with a sequence binned as “A. phosphatis”, i.e. medium strong association with the “A. phosphatis” composite genome], and red [best MASCOT sequence match located on a scaffold source binned as “other Accumulibacter” for which a weak BLAST hit (<90 % identity) was obtained with a sequence binned as “A. phosphatis”, i.e. weak association with the “A. phosphatis” composite genome]. Not all intermediate metabolites are shown. Abbreviations: ACC, acetyl-CoA carboxylase; ACD, acyl-CoA dehydrogenase; ACS, acyl-CoA synthetase; AGP, ADP-glucose pyrophosphorylase; ATPsyn, F₀F₁-type ATP synthase; CSY, citrate synthase; Fba, fructose bisphosphate aldolase; HpI, hydroxypyruvate isomerase; Ily, isocitrate lyase; Mdh, malate dehydrogenase; MalS, malate synthase; NADH, uncharacterised NAD(FAD)-dependent dehydrogenase; PhaA, acetyl-CoA acetyltransferase; PhaC, poly(3-hydroxyalkanoate) synthetase; PhaJ, enoyl-CoA hydratase; PpS, phosphoenolpyruvate synthase; Pst, ABC-type phosphate transport system; SCFA, short chain fatty acids; SuccDH, succinate dehydrogenase; TpI, triosephosphate isomerase.